Bubble-based microvalve and its use in microfluidic chip

ABSTRACT

Provided are a bubble-based microvalve and a microfluidic chip using the microvalve. Also provided are methods of using the microvalve for manipulating fluid in a microfluidic channel by changing the volume and/or location of the gas in the microvalve.

TECHNICAL FIELD

The present invention is related to the field of microfluidic systems. In particular, the invention provides a bubble-based microvalve and a microfluidic chip using this microvalve.

BACKGROUND ART

Microfluidics is a technology that deals with various chemical and biological progresses, relying on precise control and manipulation of fluids that are constrained to micro-channel network exploited on a chip based on MEMS (micro-electro-mechanical systems) technology. In the early stage, chip-based capillary electrophoresis is its main research field, structures and functions of chip are relatively simple. Recently, the demand of miniaturization and integration has become a new driving force, which thus extends research field to chemical and biological reactions, such as nucleic acid amplification, immune reaction and cell analysis. Accordingly, a number of independent and uniform reaction chambers, that is a micro-reactor array, need to be configured on a chip for investigating complex reaction parameters.

According to different sampling methods, the construction of micro-reactor array typically has two types: parallel and serial. In a parallel type micro-reactor array each chamber has a filling channel, samples enter into chamber along each filling channel in parallel manner. In order to avoid non-uniformity of different chambers, the design accuracy of structures and surface properties of materials should achieve a higher standard, these requirements are not easy to be met. By contrast, in a serial type micro-reactor array all chambers share a filling channel, samples enter into chambers along this unique filling channel in serial manner. The uniformity of different chambers is easy to control, but the independence of different chambers needs to be controlled with microvalves.

Microvalves found today include pneumatic microvalves (Unger et al. (2000) Science 288:113-116), piezoelectric microvalves, phase change microvalves, torque-actuated microvalves (Chen et al. (2009) Lab Chip 9:3511-3516) and so on. There are several disadvantages to these microvalves: they are difficult to produce and to match with portable instruments, require complicated operation, and are not friendly to ordinary users.

SUMMARY OF THE INVENTION

The present invention relates to a bubble-based microvalve and a microfluidic system and chip using such microvalve, and their methods of use. Therefore, in one aspect, provided herein is a bubble-based microvalve, which microvalve comprises a gas chamber/channel connected to a fluid channel.

In some embodiments, the gas chamber/channel may be directly connected to the fluid channel. In some embodiments, the gas chamber/channel may be connected to the fluid channel through a connecting channel, a gas-permeable membrane, a gas-permeable plate, or a gas-repellent film. In some embodiments, the microvalve may further comprise a second gas channel connected to the fluid channel. In some embodiments, the gas chamber/channel may comprise a drying material or a gas-trapping material. In some embodiments, the drying material may be selected from the group consisting of silica gel, calcium chloride, aluminum oxide and magnesium oxide. In some embodiments, the gas chamber/channel may comprise a nitrogen gas.

Also provided herein is a microfluidic reaction system comprising a microvalve, which microvalve comprises a gas chamber/channel connected to a fluid channel. In some embodiments, the microfluidic reaction system may comprise a reaction chamber. In some embodiments, the microfluidic reaction system may comprise multiple reaction chambers and multiple microvalves. In some embodiments, each microvalve may be flanked by two adjacent reaction chambers, wherein the reaction chambers may be in fluidic connection with the fluidic channel. In some embodiments, the microfluidic reaction system may be open or closed.

In some embodiments, the fluid channel may have sections of different widths. In some embodiments, the size of the connecting channel may be adjustable based on at least the following parameters: pressure of injection pump, injector pipette or other injections devices or methods, volume of gas chamber/channel, ambient temperature, ambient humidity, air humidity inside the fluidic channel, angle of the fluidic channel and the connecting channel, surface tension of the fluidic sample, and hydrophobic property of the fluidic channel. In some embodiments, the connecting channel may have a length of ≦10 cm and a width of ≦1 cm. In some embodiments, the fluid channel may have branches. In some embodiments, the gas chambers/channels may be connected by an interconnecting channel. In some embodiments, the microfluidic reaction system may further comprise a means to actuate and/or stop the microvalve.

Further provided herein is a microfluidic chip comprising a microfluidic reaction system described herein. In some embodiments, the microfluidic chip may further comprise a heating device capable of heating the gas chamber/channel. In some embodiments, the heating device may comprise a resistance wire, a resistance film or a metal particle. In some embodiments, the metal particle may be a gold nano-particle. In some embodiments, the microfluidic reaction system may further comprise a cooling device capable of cooling the gas chamber/channel. In some embodiments, the cooling device may comprise a cooling fluid.

In some embodiments, the microfluidic chip may comprise a top layer and a bottom layer. In some embodiments, the top layer may contain the microfluidic reaction system. In some embodiments, the bottom layer may comprise the heating device or cooling device. In some embodiments, the top layer may contain the fluidic channel and the bottom layer may contain the gas chamber/channel. In some embodiments, the microfluidic chip further may comprise a gas-permeable membrane, a gas-permeable plate, or a gas-repellent film. In some embodiments, the pore size of the gas-permeable membrane ranges from about 1 nm to about 1 mm. In some embodiments, the material of the gas-permeable membrane may be a polymer, which may be selected from the group consisting of cellulose, cellulose acetate, cellulose nitrate, mixed cellulose, polyolefin, polyimide, polyamide, polyether sulfone, polyethylene glycol, sodium alginate, chitin, and silicone polymer. In some embodiments, the silicone polymer may be polydimethylsiloxane. In some embodiments, the gas-permeable plate may comprise pores used for connecting the gas chamber/channel. In some embodiments, the height of the pores may be not more than 10 cm, and the diameter of the pores may be not more than 1 cm. In some embodiments, the material of the gas-permeable plate may be selected from the group consisting of metal, glass, quartz, silicon, ceramic, plastic, rubber, aluminosilicate and a composite/compound thereof. In some embodiments, the material of the gas-repellent film may be selected from the group consisting of metal, glass, quartz, silicon, ceramic, plastic, rubber and a composite/compound thereof. In some embodiments, the microfluidic chip may further comprise an interconnecting channel capable of connecting the gas chambers/channels. In some embodiments, the interconnecting channel may comprise gas, liquid or mixture of gas and liquid. In some embodiments, the material of the microvalve may be selected from the group consisting of metal, glass, quartz, silicon, ceramic, plastic, rubber, aluminosilicate and a composite/compound thereof.

In another aspect, the present invention provides a method for manipulating fluid in a microfluidic channel using a microvalve, which microvalve comprises a gas chamber/channel connected to a fluid channel, wherein the volume and/or location of the gas in the microvalve is changed.

In some embodiments, the microvalve may be actuated by heating. In some embodiments, the gas chamber/channel may be placed in a waterbath or in close proximity to a heater, optionally a Pt electrode. In some embodiments, the microvalve may be actuated by cooling, wherein the cooling may be affected by injecting a cooling fluid into a channel in close proximity to the gas chamber/channel. In some embodiments, the microvalve may be actuated by adding substance, such as nitrogen, into the gas chamber/channel, wherein the substance may be gas, liquid or solid. In some embodiments, the gas may be from outside or inside of the fluidic channel, the microfluidic system or the microfluidic chip. In some embodiments, the gas from inside of the fluidic channel may be generated by a physical, electrochemical or chemical method. In some embodiments, the microvalve may be actuated by removing a substance from the gas chamber/channel. In some embodiments, the substance may be removed using the second gas channel. In some embodiments, the microvalve may be actuated by exerting force on the gas chamber/channel, wherein the force leads to deformation of the gas chamber/channel. In some embodiments, the microvalve may be actuated by a low-humidity gas.

Further provided herein is a use of a microfluidic chip described herein for a chemical or biological reaction. In some embodiments, the biological reaction may be nucleic acid amplification, immune reaction such as immunoassays, or cell analysis such as cell culture or lysis, wherein the nucleic acid amplification may be selected from the group consisting of polymerase chain reaction (PCR), strand displacement amplification (SDA), ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA) and helicase-dependent amplification (HDA).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of an embodiment of the microvalve (the microvalve is not actuated).

FIG. 2 is a schematic illustration of an embodiment of the microvalve (the microvalve is actuated).

FIG. 3 schematically illustrates the serial construction of micro-reactor array (the microvalve is not actuated).

FIG. 4 is a schematic illustration of an embodiment of the microvalve in Example 1 (the microvalve is not actuated).

FIG. 5 is a schematic illustration of an embodiment of the microvalve in Example 1 (the microvalve is actuated).

FIG. 6 schematically illustrates the location of drying material in Example 3.

FIG. 7 is a schematic illustration of an embodiment of the microfluidic chip in Example 2.

FIG. 8 is a schematic illustration of an embodiment of the microfluidic chip in Example 5.

FIG. 9 is a schematic illustration of an embodiment of the microfluidic chip in Example 6.

FIG. 10 is a schematic illustration of an embodiment of the microfluidic chip in Example 7.

FIG. 11 is a schematic illustration of an embodiment of the microfluidic chip in Example 8.

FIG. 12 is a schematic illustration of an embodiment of the microvalve in Example 9 (the microvalve is actuated).

FIG. 13 is a schematic illustration of an embodiment of the microvalve in Example 10 (the microvalve is not actuated).

FIG. 14 is a schematic illustration of an embodiment of the microvalve in Example 10 (the microvalve is actuated).

FIG. 15 is a schematic illustration of an embodiment of the microvalve in Example 10 (the microvalve is actuated again).

DETAILED DESCRIPTION OF THE INVENTION

One object of the present invention is to provide a bubble-based microvalve, which could control the flow of fluids efficiently and conveniently, and ensure the independence of different chambers of a micro-reactor array.

A. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, applications, published applications and other publications referred to herein are incorporated by reference in their entirety. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference.

As used herein, the singular forms “a”, “an”, and “the” include plural references unless indicated otherwise. For example, “a” dimer includes one or more dimers.

As used herein, the term “microfluidic device” generally refers to a device through which materials, particularly fluid borne materials, such as liquids, can be transported, in some embodiments on a micro-scale, and in some embodiments on a nanoscale. Thus, the microfluidic devices described by the presently disclosed subject matter can comprise microscale features, nanoscale features, and combinations thereof.

Accordingly, an exemplary microfluidic device typically comprises structural or functional features dimensioned on the order of a millimeter-scale or less, which are capable of manipulating a fluid at a flow rate on the order of a μL/min or less. Typically, such features include, but are not limited to channels, fluid reservoirs, reaction chambers, mixing chambers, and separation regions. In some examples, the channels include at least one cross-sectional dimension that is in a range of from about 0.1 μm to about 500 μm. The use of dimensions on this order allows the incorporation of a greater number of channels in a smaller area, and utilizes smaller volumes of fluids.

A microfluidic device can exist alone or can be a part of a microfluidic system which, for example and without limitation, can include: pumps for introducing fluids, e.g., samples, reagents, buffers and the like, into the system and/or through the system; detection equipment or systems; data storage systems; and control systems for controlling fluid transport and/or direction within the device, monitoring and controlling environmental conditions to which fluids in the device are subjected, e.g., temperature, current, and the like.

As used herein, the terms “channel,” “micro-channel,” “fluidic channel,” and “microfluidic channel” are used interchangeably and can mean a recess or cavity formed in a material by imparting a pattern from a patterned substrate into a material or by any suitable material removing technique, or can mean a recess or cavity in combination with any suitable fluid-conducting structure mounted in the recess or cavity, such as a tube, capillary, or the like.

As used herein, the terms “flow channel” and “control channel” are used interchangeably and can mean a channel in a microfluidic device in which a material, such as a fluid, e.g., a gas or a liquid, can flow through. More particularly, the term “flow channel” refers to a channel in which a material of interest, e.g., a solvent or a chemical reagent, can flow through. Further, the term “control channel” refers to a flow channel in which a material, such as a fluid, e.g., a gas or a liquid, can flow through in such a way to actuate a valve or pump.

As used herein, “chip” refers to a solid substrate with a plurality of one-, two- or three-dimensional micro structures or micro-scale structures on which certain processes, such as physical, chemical, biological, biophysical or biochemical processes, etc., can be carried out. The micro structures or micro-scale structures such as, channels and wells, electrode elements, electromagnetic elements, are incorporated into, fabricated on or otherwise attached to the substrate for facilitating physical, biophysical, biological, biochemical, chemical reactions or processes on the chip. The chip may be thin in one dimension and may have various shapes in other dimensions, for example, a rectangle, a circle, an ellipse, or other irregular shapes. The size of the major surface of chips of the present invention can vary considerably, e.g., from about 1 mm² to about 0.25 m². Preferably, the size of the chips is from about 4 mm² to about 25 cm² with a characteristic dimension from about 1 mm to about 5 cm. The chip surfaces may be flat, or not flat. The chips with non-flat surfaces may include channels or wells fabricated on the surfaces.

It is understood that aspects and embodiments of the invention described herein include “consisting” and/or “consisting essentially of” aspects and embodiments.

Other objects, advantages and features of the present invention will become apparent from the following specification taken in conjunction with the accompanying drawings.

B. Bubble-Based Microvalve, Microfluidic System and Chip

In one aspect, the present invention provides a bubble-based microvalve and a microfluidic system and chip using such microvalve, which comprises a gas chamber/channel connected to a fluid channel. According to some embodiments, the gas chamber/channel may be directly connected to the fluid channel. In some embodiments, the gas chamber/channel may be connected to the fluid channel through a connecting channel, a gas-permeable membrane, a gas-permeable plate, or a gas-repellent film. In some embodiments, the microvalve may further comprise a second gas channel connected to the fluid channel. In some embodiments, the gas chamber/channel may comprise a drying material or a gas-trapping material. In some embodiments, the drying material or the gas-trapping material may be selected from the group consisting of silica gel, calcium chloride, aluminum oxide and magnesium oxide. In some embodiments, the gas chamber/channel may comprise a nitrogen gas. In some embodiments, the material of the microvalve may be selected from the group consisting of metal, glass, quartz, silicon, ceramic, plastic, rubber, aluminosilicate and a composite/compound thereof. The gas chamber/channel may have any shape that is suitable for its function.

The microvalve may not only be suitable for liquid samples, but also for solid-liquid mixed samples. The liquid samples may have a high surface tension coefficient (e.g., water), or a lower coefficient, such as an enzyme reaction solution, a nucleic acid amplification system solution, or a sodium dodecyl sulfate (SDS) solution.

Also provided herein is a microfluidic reaction system comprising a microvalve, which microvalve comprises a gas chamber/channel connected to a fluid channel. In some embodiments, the microfluidic reaction system may comprise a reaction chamber. In some embodiments, the microfluidic reaction system may comprise multiple reaction chambers and multiple microvalves. According to some embodiments, the gas chambers and/or said gas channels may not be linked to one another. According to some embodiments, the gas chambers and/or said gas channels may be interconnected through an interconnecting channel. In some embodiments, each microvalve may be flanked by two adjacent reaction chambers, wherein the reaction chambers may be in fluidic connection with the fluidic channel. In some embodiments, the microfluidic reaction system may be open or closed. Opening and closing of the system may be controlled by the sealing of the outlet(s) of the fluidic channel.

In some embodiments, the fluid channel may have sections of different widths. In some embodiments, the size of the connecting channel may be adjustable based on at least the following parameters: pressure of injection pump or injector pipette, volume of gas chamber/channel, ambient temperature, ambient humidity, air humidity inside the fluidic channel, angle of the fluidic channel and the connecting channel, surface tension of the fluidic sample, and hydrophobic property of the fluidic channel. In some embodiments, the connecting channel may have a length of ≦10 cm and a width of ≦1 cm. In some embodiments, the fluid channel may have branches. In some embodiments, the gas chambers/channels are connected by an interconnecting channel.

In some embodiments, the microfluidic reaction system may further comprise a means to actuate and/or stop the microvalve. Any suitable means to actuate and/or stop the bubble-based microvalve may be used, e.g., introducing a substance into the microvalve or exerting force to the microvalve, wherein the substance may be a gas, liquid or solid substance, and wherein the force may be a piezoelectric, electric, pneumatic or magnetic force.

Exemplary actuation means of the microvalve may be: 1) low-humidity gas, wherein the low-humidity gas may be atmospheric air or gas trapped in the microfluidic system, and the latter may be produced by a drying material, such as silica gel, calcium chloride, aluminum oxide or magnesium oxide; 2) heating, wherein the object of heating may be the microvalve or the whole microfluidic system, and heating methods may relate to heat conduction, resistance, electromagnetic, ultrasonic, laser or infrared; 3) cooling, wherein the object of cooling may be microvalve or the whole microfluidic system, and the cooling medium may be water, air or oil; 4) adding a substance, wherein the substance may be gas, liquid or solid; 5) removing a substance, wherein the substance may be gas, liquid or solid; and 6) exerting a force, wherein the force may deform the gas chamber/channel or change the internal pressure, which may lead to bubble generation/removal. These actuation means disclosed herein may be used alone or in combination.

In some embodiments, outside gas may be introduced into the microfluidic system. In some embodiments, inside gas may be introduced into the gas chamber and/or gas channel. The inside gas may be stored in the microfluidic system already, or may be new gas generated though, for example, physical method (e.g., heating of solid or liquid regents), electrochemical method (e.g., electrolysis of salt solution), or chemical method (e.g., acid-base reaction). In some embodiments, a substance outside may be removed to induce a low pressure, which thus leads to bubble because of gas expansion. In some embodiments, forces can be piezoelectric, electric, pneumatic or magnetic. The object exerting the forces may be the gas chamber/gas channel or other parts of the microfluidic system, and the device exerting the forces may be outside or inside the microfluidic system.

In some embodiments, the microfluidic reaction system may further comprise a means to stop the microvalve. Generally the stop means may be the reverse operation of the actuation means, e.g., stop heating, stop cooling, remove filled substances, or stop exerting the force. Other stop means may be directly manipulating the bubble, e.g., extracting bubble with a syringe needle, or reducing bubble volume by a chemical or biological reaction. These stop means can be used alone or in combination.

Further provided herein is a microfluidic chip comprising a microfluidic reaction system described herein. In some embodiments, the microfluidic chip may further comprise a heating device capable of heating the gas chamber/channel. In some embodiments, the heating device may comprise a resistance wire, a resistance film or a metal particle. In some embodiments, the metal particle may be a gold nano-particle. Any suitable heating means may be used, such as heat conduction, electromagnetic, ultrasonic, laser or infrared. In some embodiments, the microfluidic reaction system may further comprise a cooling device capable of cooling the gas chamber/channel. In some embodiments, the cooling device may comprise a cooling fluid.

In some embodiments, the microfluidic chip may comprise a top layer and a bottom layer. In some embodiments, the top layer may contain the microfluidic reaction system. In some embodiments, the bottom layer may comprise the heating device or cooling device. In some embodiments, the top layer may contain the fluidic channel and the bottom layer may contain the gas chamber/channel. In some embodiments, the microfluidic chip further may comprise a gas-permeable membrane, a gas-permeable plate, or a gas-repellent film. In some embodiments, the pore size of the gas-permeable membrane ranges from about 1 nm to about 1 mm. In some embodiments, the material of the gas-permeable membrane may be a polymer, which may be selected from the group consisting of cellulose, cellulose acetate, cellulose nitrate, mixed cellulose, polyolefin, polyimide, polyamide, polyether sulfone, polyethylene glycol, sodium alginate, chitin, and silicone polymer. In some embodiments, the silicone polymer may be polydimethylsiloxane. In some embodiments, the gas-permeable plate may comprise pores used for connecting the gas chamber/channel. In some embodiments, the height of the pores may be not more than 10 cm, and the diameter of the pores may be not more than 1 cm. In some embodiments, the material of the gas-permeable plate may be selected from the group consisting of metal, glass, quartz, silicon, ceramic, plastic, rubber, aluminosilicate and a composite/compound thereof. In some embodiments, the material of the gas-repellent film may be selected from the group consisting of metal, glass, quartz, silicon, ceramic, plastic, rubber and a composite/compound thereof. In some embodiments, the microfluidic chip may further comprise an interconnecting channel capable of connecting the gas chambers/channels. In some embodiments, the interconnecting channel may comprise gas, liquid or mixture of gas and liquid.

In order to prevent a fluidic sample from entering into gas chamber and/or gas channel, the size of the connecting channel (width/height/length, etc.) may be adjusted based on at least the following parameters: pressure of injection pump or injector pipette, volume of gas chamber and/or gas channel, ambient temperature, ambient humidity, air humidity inside the fluidic channel, angle of the fluidic channel and the connecting channel, surface tension of the fluidic sample, and hydrophobic property of the fluidic channel. The length and width of the connecting channel may be set according to actual needs, for example, the connecting channel may have a length of ≦10 cm, and a width of ≦1 cm.

According to some embodiments, the gas chamber/channel may be connected to the fluid channel through a connecting channel, a gas-permeable membrane, a gas-permeable plate, or a gas-repellent film. According to some embodiments, a gas-permeable membrane may be located between the gas chamber and/or gas channel and the fluidic channel. This may prevent the fluidic sample from entering into gas chamber and/or gas channel, and may ensure the connection between the gas chamber and/or gas channel and the fluidic channel in the meanwhile. The pore size of the gas-permeable membrane may be adjusted according to actual needs, for example, the pore size may range from about 1 nm to about 1 mm. According to some embodiments, a gas-permeable plate may be located between the gas chamber and/or gas channel and the fluidic channel. The role of gas-permeable plate may be similar to the gas-permeable membrane. The length and diameter of the pores of the gas-permeable plate may be set according to actual needs, for example, length may be ≦10 cm, diameter may be ≦1 cm. In some embodiments, the diameter may be 10 nm. According to some embodiments, a gas-repellent film may be located between the gas chamber and/or gas channel and the fluidic channel, wherein the gas-repellent film may be used to actuate the microvalve in some cases. The material of the gas-permeable plate, gas-permeable membrane and gas-repellent film may be selected from the group consisting of metal, glass, quartz, silicon, ceramic, plastic, rubber, aluminosilicate, and a composite/compound thereof.

According to some embodiments, a connecting channel, a gas-permeable membrane, a gas-permeable plate, and a gas-repellent film may also be used in combination, and the spatial relationship between the gas chamber/channel and the connecting channel, gas-permeable membrane, gas-permeable plate, and gas-repellent film is not limited.

The microfluidic devices of the present invention may comprise a central body structure in which various microfluidic elements are disposed. The body structure includes an exterior portion or surface, as well as an interior portion which defines the various microscale channels and/or chambers of the overall microfluidic device. For example, the body structure of the microfluidic devices of the present invention typically employs a solid or semi-solid substrate that may be planar in structure, i.e., substantially flat or having at least one flat surface. Suitable substrates may be fabricated from any one of a variety of materials, or combinations of materials. Often, the planar substrates are manufactured using solid substrates common in the fields of microfabrication, e.g., silica-based substrates, such as glass, quartz, silicon or polysilicon, as well as other known substrates, i.e., gallium arsenide. In the case of these substrates, common microfabrication techniques, such as photolithographic techniques, wet chemical etching, micromachining, i.e., drilling, milling and the like, may be readily applied in the fabrication of microfluidic devices and substrates. Alternatively, polymeric substrate materials may be used to fabricate the devices of the present invention, including, e.g., polydimethylsiloxanes (PDMS), polymethylmethacrylate (PMMA), polyurethane, polyvinylchloride (PVC), polystyrene, polysulfone, polycarbonate and the like. In the case of such polymeric materials, injection molding or embossing methods may be used to form the substrates having the channel and reservoir geometries as described herein. In such cases, original molds may be fabricated using any of the above described materials and methods.

The channels and chambers of the device are typically fabricated into one surface of a planar substrate, as grooves, wells or depressions in that surface. A second planar substrate, typically prepared from the same or similar material, is overlaid and bound to the first, thereby defining and sealing the channels and/or chambers of the device. Together, the upper surface of the first substrate, and the lower mated surface of the upper substrate, define the interior portion of the device, i.e., defining the channels and chambers of the device. In some embodiments, the upper layer may be reversibly bound to the lower layer.

In the exemplary devices described herein, at least one main channel, also termed an analysis channel, is disposed in the surface of the substrate through which samples are transported and subjected to a particular analysis. Typically, a number of samples are serially transported from their respective sources, and injected into the main channel by placing the sample in a transverse channel that intersects the main channel. This channel is also termed a “sample loading channel.” The sample sources are preferably integrated into the device, e.g., as a plurality of wells disposed within the device and in fluid communication with the sample loading channel, e.g., by an intermediate sample channel.

The systems of the invention may also include sample sources that are external to the body of the device per se, but still in fluid communication with the sample loading channel. In some embodiments, the system may further comprise an inlet and/or an outlet to the micro-channel. In some embodiments, the system may further comprise a delivering means to introduce a sample to the micro-channel. In some embodiments, the system may further comprise an injecting means to introduce a liquid into the micro-channel. Any liquid manipulating equipments, such as pipettes, pumps, etc., may be used as an injecting means to introduce a liquid to the micro-channel.

C. Methods of Manipulating Fluid Using the Microvalve

In another aspect, the present invention provides a method for manipulating fluid in a microfluidic channel using a microvalve, which microvalve comprises a gas chamber/channel connected to a fluid channel, wherein the volume and/or location of the gas in the microvalve is changed.

In some embodiments, the microvalve may be actuated by heating. In some embodiments, the gas chamber/channel may be placed in a waterbath or in close proximity to a heater, optionally a Pt electrode. In some embodiments, the microvalve may be actuated by cooling, wherein the cooling may be by injecting a cooling fluid into a channel in close proximity to the gas chamber/channel. In some embodiments, the microvalve may be actuated by adding substance, such as nitrogen, into the gas chamber/channel, wherein the substance may be gas, liquid or solid. In some embodiments, the gas may be from outside or inside of the fluidic channel, the microfluidic system or the microfluidic chip. In some embodiments, the gas from inside of the fluidic channel may be generated by a physical, electrochemical or chemical method. In some embodiments, the microvalve may be actuated by removing a substance from the gas chamber/channel. In some embodiments, the substance may be removed using the second gas channel. In some embodiments, the microvalve may be actuated by exerting force on the gas chamber/channel, wherein the force leads to deformation of the gas chamber/channel. In some embodiments, the microvalve may be actuated by a low-humidity gas.

Further provided herein is a use of a microfluidic chip described herein for a chemical or biological reaction. In some embodiments, the biological reaction may be nucleic acid amplification, immune reaction or cell analysis such as cell culture or lysis, wherein the nucleic acid amplification may be selected from the group consisting of polymerase chain reaction (PCR), strand displacement amplification (SDA), ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA) and helicase-dependent amplification (HDA).

D. Examples

The following examples are offered to illustrate but not to limit the invention.

REFERENCE NUMERAL LIST

-   101 fluidic channel -   102 connecting channel -   103 gas chamber -   201 sample -   202 gas -   301 bubble-based microvalve -   302 reaction chamber -   401 narrow branch channel -   402 wide branch channel -   403 necked channel -   701 silica gel bead -   801 Pt electrodes -   901 cooling channel -   1001 interconnecting channel -   1002 gas outlet -   1101 pyramid -   1201 channel-bubble-based microvalve -   1201 gas channel -   1301 combined bubble-based microvalve

EXAMPLE 1 Low-Humidity Environment, Open System (Outlet is Not Sealed After Injection), the Microvalve is Actuated by Low-Humidity Air

The device is composed of two layers, a 1 mm thick PMMA cover layer and a 2 mm thick PMMA bottom layer. The bottom layer is embedded with one channel (101) (w=0.5 mm), one narrow branch channel (401) (w=0.2 mm) connecting the channel (101), and one wide branch channel (402) (w=0.5 mm) connecting the channel (101) through a necked channel (403) (w=0.2 mm). It further contains the bubble-based microvalve (301), in which a gas chamber (103) (l×w×h=2.2 mm×0.5 mm×1.5 mm) with a hydrophobic Teflon (PTFE) coating is directly connected to the channel (101). All channels are 0.2 mm deep.

The device is fabricated through conventional techniques in the microfluidic area, i.e., firstly micro-structures on the PMMA bottom layer are exploited by laser engraving machine or milling machine, then a PTFE solution (0.1% V/V) is coated into gas chamber using a injector pipette, and finally the cover layer and the bottom layer are thermally bonded into a complete chip.

The device is positioned in the lab with room temperature 20° C., relative humidity 16%. When injecting water sample to channel (101) in 5 μL/min, most of the sample is entering into the wide branch channel (402) because of the bigger fluid resistance of narrow branch channel (401) (FIG. 3). Three minutes later, the volume increase of gas chamber (202) induces a bubble into the channel (101), thus held by the necked channel (403), so the wide branch channel (402) is blocked while sample can only enter into narrow branch channel (401) (FIG. 2, 5).

The working principle is: since PTFE coating is hydrophobic and water sample has a high surface tension, water does not fill the gas chamber (103) and leaving trapped low-humidity air inside the gas chamber (103). After priming, a part of sample (201) will spontaneously evaporate into gas (202) to increase the humidity and pressure, which generate a bubble to block the downstream flow.

EXAMPLE 2 Low-Humidity Environment, Closed System (Outlet is Sealed After Injection), the Microvalve is Actuated by Low-Humidity Air

The microfluidic chip is composed of two layers, a 1 mm thick PMMA cover layer and a 2 mm thick PMMA bottom layer. The bottom layer is embedded with a micro-reactor array, in which a channel (101) and 24 reaction chambers (302) are arranged serially in a ring pattern and the distance between each chamber (diameter=3 mm, depth=1 mm) is uniform (FIG. 6). It further contains the bubble-based microvalve (301), in which each gas chamber (103) (diameter=1.8 mm, depth=1.5 mm) between reaction chambers (302) is connected to the channel (101) through a connecting channel (102) (l=0.75 mm). All channels are 0.2 mm deep, 0.4 mm wide.

The fabrication technology of this device is similar to Example 2.

The device is positioned in the lab with room temperature 20° C., relative humidity 16%. When injecting PCR system solution (201) to channel (101) in 60 μL/min, this sample serially enters into each reaction chamber (302), then the inlet and outlet are sealed. Three minutes later, the volume increase of gas chamber (202) induces a bubble into the channel (101), thus blocking the channel (101) and isolating the reaction chamber (302).

The working principle is: since fluid pressure is relatively high and PCR system solution has a low surface tension, a connecting channel (102) is necessary to prevent the sample from filling the gas chamber (103). After priming, a part of sample (201) will spontaneously evaporate into gas (202) to increase the humidity and pressure, which generate a bubble to isolate the reaction chamber (302).

EXAMPLE 3 High-Humidity Environment, Closed System (Outlet is Sealed after Injection), the Microvalve is Actuated by Low-Humidity Gas Trapped in Chip

The microfluidic chip is the same as the one in Example 2, except that each gas chamber (103) contains a silica gel bead (701) (FIG. 7).

The device is positioned in the lab with room temperature 20° C., relative humidity 75%. Silica gel beads (701) are added into each chamber and the inlet and outlet are sealed for one hour. After that, a PCR system solution (201) is injected to the channel (101) in 60 μL/min, then the inlet and outlet are sealed once again. Three minutes later, the volume increase of gas chamber (202) induces a bubble into the channel (101), thus blocking the channel (101) and isolating the reaction chamber (302).

The working principle is: the principle is the same as the one in Example 2, except that the low-humidity air is instead of low-humidity gas dried by silica gel beads.

EXAMPLE 4 High-Humidity Environment, Closed System (Outlet is Sealed after Injection), the Microvalve is Actuated through Heating the Whole Microfluidic Chip

The microfluidic chip is the same as the one in Example 2.

The device is positioned in the lab with room temperature 20° C., relative humidity 75%. When injecting PCR system solution (201) to channel (101) in 60 μL/min, this sample serially enters into each reaction chamber (302), then the inlet and outlet are sealed. The chip is placed into the thermostat water bath at 65° C., which raises the temperature of the whole microfluidic chip through heat conduction. Two minutes later, the volume increase of gas chamber (202) induces a bubble into the channel (101), thus blocking the channel (101) and isolating the reaction chamber (302) (FIG. 2).

The working principle is: because of heating, a part of sample (201) will evaporate into gas (202), which raises the saturation vapor pressure and reduces the liquid volume, thus a bubble is generated to isolate the reaction chamber (302). It is should be noted that the chip are sealed in the progress, thermal expansion of the gas does not increase the gas volume, but increase the pressure.

EXAMPLE 5 High-Humidity Environment, Open System (Outlet is Not Sealed after Injection), the Microvalve is Actuated through Heating the Gas Chamber

As shown in FIG. 8, the microfluidic chip is composed of two layers, a 4 mm thick PDMS cover layer and a 2 mm thick glass bottom layer (the dashed line in the bottom layer represents the projection of cover layer). The cover layer is embedded with a micro-reactor array, in which a channel (101) and 42 reaction chambers (302) (diameter=3 mm, depth=1 mm) are arranged serially in a snake pattern and the distance between each reaction chamber (diameter=4 mm, depth=1 mm) is uniform. It further contains the bubble-based microvalve (301), in which each gas chamber (103) (l×w×h=3 mm×3 mm×1.5 mm) between reaction chambers (302) is connected to the channel (101) through a connecting channel (102) (l=1 mm). The bottom layer contains a heater, Pt meander traces (801) aligned with gas chambers (103). All channels are 0.2 mm deep, 0.2 mm wide.

The cover layer of the microfluidic chip is fabricated in polydimethylsiloxane (PDMS) using a rapid prototyping technique and Pt electrodes of the bottom layer are lithographically patterned onto a glass slide. Pt electrodes (801) are connected to an external power for heating gas chambers (103) alone, that will avoid thermal denaturation of sample in reaction chambers (302).

The device is positioned in the lab with room temperature 20° C., relative humidity 75%. When injecting SDS solution (201) (10% W/V) to channel (101) at 360 μL/min, this sample serially enters into each reaction chamber (302), the inlet and outlet are not sealed. Gas chambers (103) are heated to 70° C. by Pt electrodes (801) while the pressure of gas chamber (202) is rising. Two minutes later, the volume increase of gas chamber (202) induces a bubble into the channel (101), thus blocking the channel (101) and isolating the reaction chamber (302) (FIG. 2).

The working principle is: since flow rate is higher than the ones in Examples 2-4, and SDS solution has a lower surface tension than PCR system solution, the connecting channel (102) should be longer and narrower to prevent the sample from filling the gas chamber (103). After heating, firstly the gas is performing pure thermal expansion, then a part of sample (201) will gradually evaporate into gas (202), which raises the saturation vapor pressure. Finally these two factors contribute to the gas volume increase, a bubble is generated to isolate the reaction chamber (302). It is should be noted that the chip is not sealed in the progress.

EXAMPLE 6 High-Humidity Environment, Closed System (Outlet is Sealed after Injection), the Microvalve is Actuated through Cooling the Gas Chamber

As shown in FIG. 9, the microfluidic chip is similar to the one in Example 5, but it is composed of three layers, a 4 mm thick PDMS cover layer, a 0.2 mm thick glass middle layer and a 2 mm thick glass bottom layer (the dashed line in the bottom layer represents the projection of cover layer). The cover layer is embedded with a micro-reactor array, in which a channel (101) and 42 reaction chambers (302) (diameter=3 mm, depth=1 mm) are arranged serially in a snake pattern and the distance between each reaction chamber (diameter=4 mm, depth=1 mm) is uniform. It further contains the bubble-based microvalve (301), in which each gas chamber (103) (l×w×h=3 mm×3 mm×1.5 mm) between reaction chambers (302) is connected to the channel (101) through a connecting channel (102) (l=1 mm). The bottom layer contains a cooling channel (901) (w×h=1 mm×0.2 mm), which is aligned with gas chambers (103). All channels are 0.2 mm deep, 0.2 mm wide.

The cover layer of the microfluidic chip is fabricated in polydimethylsiloxane (PDMS) using a rapid prototyping technique and the cooling channel (901) in the bottom layer are patterned onto a glass slide using a wet etching method. The cooling channel (901) is filled with cooling solution by a peristaltic pump, that can only cool gas chambers (103) and not affect reaction chambers (302).

The device is positioned in the lab with room temperature 20° C., relative humidity 75%. When injecting SDS solution (201) (10% W/V) to channel (101) at 360 μL/min, this sample serially enters into each reaction chamber (302), then the inlet and outlet are sealed. Saline solution at 0° C. is injected to the cooling channel (901) at 1 mL/min. One minute later, a bubble is induced into the channel (101), thus blocking the channel (101) and isolating the reaction chamber (302) (FIG. 2).

The working principle is: after cooling, gas chambers will change to near 0° C., and vapor from the relatively warmer region will condensate in the walls of cooler gas chambers, i.e., mass transfer between the liquid in the channel (101) and the gas in the gas chamber (103) is happening, a part of gas is displaced into the channel (101), and a bubble is generated to isolate the reaction chamber (302). It should be noted that the chip is sealed in the process, thermal contraction of the gas does not reduce the gas volume, but reduces the pressure.

EXAMPLE 7 High-Humidity Environment, Closed System (Outlet is Sealed after Injection), the Microvalve is Actuated through Adding Substances into the Gas Chamber

As shown in FIG. 10, the microfluidic chip is similar to the one in Example 6, it is composed of three layers, a 4 mm thick PDMS cover layer, a 0.05 mm thick PDMS middle layer and a 2 mm thick glass bottom layer (the dashed line in the bottom layer represents the projection of cover layer). The cover layer is embedded with a micro-reactor array, in which a channel (101) and 42 reaction chambers (302) (diameter=3 mm, depth=1 mm) are arranged serially in a snake pattern and the distance between each reaction chamber (diameter =4 mm, depth=1 mm) is uniform. The bottom layer contains interconnecting channels (1001) (w×h=1 mm×0.2 mm) and gas chambers (103) (w×h=1 mm×0.2 mm), which is aligned with the channel (101) of the cover layer.

Gas chambers (103) are interconnected by a few interconnecting channels (1001), and share a unique gas outlet (1002), i.e., all the bubble-based microvalves (301) are connected as a complete gas network. The cover layer of the microfluidic chip is fabricated in polydimethylsiloxane (PDMS) using a rapid prototyping technique and bubble-based microvalves (301) in the bottom layer are patterned onto a glass slide using a wet etching method.

The device is positioned in the lab with room temperature 20° C., relative humidity 75%. When injecting SDS solution (201) (10% W/V) to channel (101) at 360 μL/min, this sample serially enters into each reaction chamber (302), then the inlet and outlet are not sealed. Nitrogen is injected to the gas outlet (1002) at 1.2 MPa. Ten minute later, the volume increase of gas (202) in gas chamber (103) induces a bubble into the channel (101), thus blocking the channel (101) and isolating the reaction chamber (302). After that, the gas outlet (1002) is closed and bubbles keep stable.

The working principle is: a gas-permeable membrane, the PDMS middle layer is placed between the channel (101) and the gas chambers (103) to prevent sample from filling the gas chambers (103), so connecting channels in Examples 5-6 are no longer needed. When nitrogen is injected, it will enter into each gas chamber (103) along the gas network, then gradually permeate into the channel (101) through PDMS middle layer. After a period of time, a bubble is generated to isolate the reaction chamber (302). Because the equilibrium of gas solubility has been set up, outside gas pressure is not required to continue.

EXAMPLE 8 High-Humidity Environment, Closed System (Outlet is Sealed after Injection), the Microvalve is Actuated through Exerting Forces onto Gas Chambers

The microfluidic chip is the same as the one in Example 2, except that the 1 mm thick PMMA cover layer is instead of 0.2 mm thick PMMA membrane. A supplementary tool, the pyramid ring (FIG. 11), is used for pressing the gas chamber. It is composed of a metal ring and 23 pyramids (1101), each pyramid is aligned with the gas chamber (103) of the cover layer.

The device is positioned in the lab with room temperature 20° C., relative humidity 75%. When injecting SDS solution (201) (10% W/V) to channel (101) at 360 μL/min, this sample serially enters into each reaction chamber (302), then the inlet and outlet are sealed. When exerting forces on the pyramid ring, which is placed on the chip, a part of the gas (202) in the gas chamber (103) is pushed out. A bubble is generated to block the channel (101) and isolate the reaction chamber (302). If removing forces at this time, the bubble will retract into the gas chamber (103), and reaction chambers (302) will not be independent.

The working principle is: the PM MA membrane will deform under the external force, which changes the location of gas and induces a bubble.

EXAMPLE 9 High-Humidity Environment, Open System (Outlet is Not Sealed after Injection), the Microvalve is Actuated through Adding Substances into the Gas Chamber

As shown in FIG. 12, the microfluidic chip is the same as the one in Example 1, except the gas chamber (103) instead of the gas channel (1202), so the microvalve can be regard as channel-bubble-based microvalve (1201). The gas channel (1202) is a PVC tube (diameter=0.1 mm), one end is bonded to the channel (101) by epoxy resin and the other is connected to a syringe.

The device is positioned in the lab with room temperature 20° C., relative humidity 75%. When injecting water sample to channel (101) in 5 μL/min, most of the sample is entering into the wide branch channel (402) because of the bigger fluid resistance of narrow branch channel (401). Air is injected into the gas channel (1202) for 2 s, which induces a bubble into the channel (101). The bubble is thus held by the necked channel (403), so the wide branch channel (402) is blocked while sample can only enter into narrow branch channel (401) (FIG. 12).

The working principle is: since the gas channel (1202) is closed by syringe in the beginning, water does not fill the gas chamber (103). Once a little air is injected, a bubble is generated to block the downstream flow.

EXAMPLE 10 High-Humidity Environment, Closed System (Outlet is Sealed after Injection), the Microvalve is Actuated through Removing Substances

As shown in FIG. 13, the microfluidic chip is the same as the one in Example 1, except that the bubble-based microvalve (301) is replaced by the combined bubble-based microvalve (1301), which contains a gas chamber (103) and a gas channel (1202). The gas channel (1202) is a PVC tube (diameter=0.1 mm), one end is bonded to the channel (101) by epoxy resin and the other is connected to a syringe.

The device is positioned in the lab with room temperature 20° C., relative humidity 75%. When injecting water sample to channel (101) in 5 μL/min, most of the sample is entering into the wide branch channel (402) because of the bigger fluid resistance of narrow branch channel (401). Using the syringe, sample is extracted into the gas channel (1202) for 2 s, which reduces the pressure of the channel (101). Accordingly, the gas in gas chamber (103) expands and induces a bubble into the channel (101). The bubble is held by the necked channel (403), so the wide branch channel (402) is blocked, thus sample can only enter into narrow branch channel (401) (FIG. 14) and the gas chamber (103) is partly filled by sample.

Sample is once again extracted into the gas channel (1202) for 10 s, the bubble disappears and the wide branch channel (402) is again open.

The working principle is: if removing substances outside gas chamber, a low pressure will be generated, then the gas volume will be increasing under the low pressure, finally that induces a bubble to block the downstream flow. If extracting bubble with syringe, the microvalve will remain open. In this embodiment, the bubble-based microvalve can control the flow stage shift between open-closed.

EXAMPLE 11 Structure of Bubble-Based Microvalve and an Actuation Means

This structure utilizes gas chamber (103), which is not connected to outside, rather than gas channel (1202).

In low-humidity environment (ambient relative humidity is 5-50%), during sample (201) priming of the channel (101), geometry character of gas chamber (103) ensures that the liquid-air phase line remains in the interface between the gas chamber (103) and the channel (101), the sample (201) does not fill (or fill partly) the gas chamber (103), meanwhile, air trapped in the gas chamber (103) keeps its original property of low-humidity. At this time the microvalve is not actuated. After sample (201) priming, a part of sample (201) will spontaneously evaporate into gas (202) until gas (202) reaches saturation, which increases the pressure of the gas (202), and finally induces a bubble into the channel (101). At this time the microvalve is actuated, the channel (101) is closed by the bubble.

In the microfluidic chip, microvalves and reaction chambers are connected to each other in series. After a sample is injected into this chip, sample volume of each chamber is uniform, and each reaction chamber can soon be independent in the action of microvalve.

The above examples are included for illustrative purposes only and are not intended to limit the scope of the invention. Many variations to those described above are possible. Since modifications and variations to the examples described above will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims.

The advantage of the microvalve described herein includes simple design, controllable operation, broad application range especially for the case that heat effect should not be introduced and the case that closed system should be ensured. 

1. A bubble-based microvalve, which microvalve comprises a gas chamber/channel connected to a fluid channel.
 2. The microvalve of claim 1, wherein the gas chamber/channel is directly connected to the fluid channel.
 3. The microvalve of claim 1, wherein the gas chamber/channel is indirectly connected to the fluid channel through a connecting channel, a gas-permeable membrane, a gas-permeable plate, or a gas-repellent film.
 4. The microvalve of claim 1, further comprising a second gas chamber/channel connected to the fluid channel.
 5. The microvalve of claim 1, wherein the gas chamber/channel comprises a drying material, and the drying material is selected from the group consisting of silica gel, calcium chloride, aluminum oxide and magnesium oxide. 6-7. (canceled)
 8. A microfluidic reaction system comprising a microvalve of claim
 1. 9. The microfluidic reaction system of claim 8, further comprising a reaction chamber.
 10. The microfluidic reaction system of claim 9, which comprises multiple reaction chambers and multiple microvalves.
 11. The microfluidic reaction system of claim 10, wherein each microvalve is flanked by two adjacent reaction chambers, and wherein the reaction chambers are in fluidic connection with the fluidic channel. 12-13. (canceled)
 14. The microfluidic reaction system of claim 8, wherein the size of the connecting channel is adjustable based on at least the following parameters: pressure of injection pump or injector pipette, volume of gas chamber/channel, ambient temperature, ambient humidity, air humidity inside the fluidic channel, angle of the fluidic channel and the connecting channel, surface tension of the fluidic sample, and hydrophobic property of the fluidic channel. 15-16. (canceled)
 17. The microfluidic reaction system of claim 8, wherein the gas chambers/channels are connected by an interconnecting channel.
 18. The microfluidic reaction system of claim 17, further comprising a means to actuate and/or stop the microvalve.
 19. A microfluidic chip comprising a microfluidic reaction system of claim
 8. 20. The microfluidic chip of claim 19, further comprising a heating device capable of heating the gas chamber/channel, wherein the heating device comprises a resistance wire, a resistance film or a metal particle. 21-22. (canceled)
 23. The microfluidic chip of claim 19, further comprising a cooling device capable of cooling the gas chamber/channel, wherein the cooling device comprises a cooling fluid.
 24. (canceled)
 25. The microfluidic chip of claim 19, wherein the microfluidic chip comprises a top layer and a bottom layer. 26-27. (canceled)
 28. The microfluidic chip of claim 25, wherein the top layer contains the fluidic channel and the bottom layer contains the gas chamber/channel.
 29. The microfluidic chip of claim 25, wherein the microfluidic chip further comprises a gas-permeable membrane, a gas-permeable plate, or a gas-repellent film. 30-33. (canceled)
 34. The microfluidic chip of claim 29, wherein the gas-permeable plate comprises pores used for connecting the gas chamber/channel. 35-37. (canceled)
 38. The microfluidic chip of claim 19, further comprising an interconnecting channel capable of connecting the gas chambers/channels, wherein the interconnecting channel comprises gas, liquid or mixture of gas and liquid. 39-40. (canceled)
 41. A method for manipulating fluid in a microfluidic channel using a microvalve of claim 1, wherein the volume and/or location of the gas in the microvalve is changed.
 42. The method of claim 41, wherein the microvalve is actuated by heating, and the gas chamber/channel is placed in a waterbath or in close proximity to a heater, optionally a Pt electrode.
 43. (canceled)
 44. The method of claim 41, wherein the microvalve is actuated by cooling, and the cooling is by injecting a cooling fluid into a channel in close proximity to the gas chamber/channel.
 45. (canceled)
 46. The method of claim 41, wherein the microvalve is actuated by adding a substance, such as gas, liquid or solid, into the gas chamber/channel. 47-49. (canceled)
 50. The method of claim 41, wherein the microvalve is actuated by removing a substance from the gas chamber/channel, and the substance is removed using the second gas channel.
 51. (canceled)
 52. The method of claim 41, wherein the microvalve is actuated by exerting force on the gas chamber/channel, and the force leads to deformation of the gas chamber/channel.
 53. (canceled)
 54. The method of claim 41, wherein the microvalve is actuated by a low-humidity gas. 55-59. (canceled) 